Pichia kudriavzevii (Candida krusei): A systematic review to inform the World Health Organisation priority list of fungal pathogens

Abstract In response to the growing global threat of fungal infections, in 2020 the World Health Organisation (WHO) established an Expert Group to identify priority fungi and develop the first WHO fungal priority pathogen list (FPPL). The aim of this systematic review was to evaluate the features and global impact of invasive infections caused by Pichia kudriavzevii (formerly known as Candida krusei). PubMed and Web of Science were used to identify studies published between 1 January 2011 and 18 February 2021 reporting on the criteria of mortality, morbidity (defined as hospitalisation and length of stay), drug resistance, preventability, yearly incidence, and distribution/emergence. Overall, 33 studies were evaluated. Mortality rates of up to 67% in adults were reported. Despite the intrinsic resistance of P. kudriavzevii to fluconazole with decreased susceptibility to amphotericin B, resistance (or non-wild-type rate) to other azoles and echinocandins was low, ranging between 0 and 5%. Risk factors for developing P. kudriavzevii infections included low birth weight, prior use of antibiotics/antifungals, and an underlying diagnosis of gastrointestinal disease or cancer. The incidence of infections caused by P. kudriavzevii is generally low (∼5% of all Candida-like blood isolates) and stable over the 10-year timeframe, although additional surveillance data are needed. Strategies targeting the identified risk factors for developing P. kudriavzevii infections should be developed and tested for effectiveness and feasibility of implementation. Studies presenting data on epidemiology and susceptibility of P. kudriavzevii were scarce, especially in low- and middle-income countries (LMICs). Thus, global surveillance systems are required to monitor the incidence, susceptibility, and morbidity of P. kudriavzevii invasive infections to inform diagnosis and treatment. Timely species-level identification and susceptibility testing should be conducted to reduce the high mortality and limit the spread of P. kudriavzevii in healthcare facilities.


Introduction
Fungal pathogens contribute to a high burden of disease and are major threats to global health.Although the burden has not been accurately measured, crude estimates suggest they cause over 1.6 million deaths annually. 1People who are immunocompromised due to cancer, chronic lung disease, tuberculosis, HIV, organ transplantation, major abdominal surgery, or are on immunosuppressive drugs are vulnerable to serious fungal infections. 1 , 2 Despite the global concern, the allocation of research support to generate robust data from clinical and microbiological studies to, in turn, support the development of effective diagnosis and treatment strategies for fungal infections has been limited to date.Lack of comprehensive surveillance systems also leaves clinicians in an evidence vacuum, relying on sparse or anecdotal information regarding local epidemiology, antimicrobial resistance, and treatment strategies to inform clinical decision-making.
In recognition of the growing global threat of fungal pathogens, in 2020 World Health Organisation (WHO) established an Expert Group to identify priority fungi and develop the first fungal priority pathogen list (FPPL).The FPPL was developed through a wide international consultation process using a survey composed of discrete choice experiments (DCE).Individual fungal pathogens were subsequently ranked based on the results of the DCE, informed by systematic reviews.This global exercise highlighted the urgent need for prioritising research and interventions against invasive fungal infections.
Invasive fungal diseases (IFD) are associated with mortality and morbidity for hospitalised patients and increased healthcare costs. 1 , 3 , 4 Whilst Candida species were a common cause of IFD in previous decades, 2 an increasing incidence of other yeast-like fungi have been reported more recently. 3 , 45][6] Consequently, P. kudriavzevii has been selected among the fungi to rank in the FPPL of the WHO.
Despite these major concerns, limited research has been conducted to support the effective diagnosis and treatment of P. kudriavzevii infections.Whilst two recent reviews have focused on the basic science aspects of P. kudriavzevii, 4 , 7 an update of clinically relevant characteristics and global impact of P. kudriavzevii invasive infections is required.
We conducted a systematic review to (1) evaluate the features and global impact of invasive infections caused by P. kudriavzevii, and (2) determine knowledge gaps for P. kudriavzevii and identify research priorities.

Study design
A systematic review was performed as per the Preferred Reporting Items for Systematic Review and Meta-Analyses (PRISMA) guidelines.

Inclusion and exclusion criteria
The criteria used to assess features and global impact of IFD caused by P. kudriavzevii (C.krusei) were mortality, hospi-talisation, disability, antifungal drug resistance, preventability, yearly incidence, global distribution, and emergence in the last 10 years.To ensure a comprehensive analysis, the chosen criteria encompass various aspects of disease burden and epidemiology.Studies were considered for inclusion if they satisfied the following criteria: (1) patient population included adults and/or paediatric patients, (2) included data on P. kudriavzevii, (3) included data on at least one criterion for the prioritisation (i.e., study measure), (4) were retrospective or prospective observational studies, randomised controlled trials, epidemiology or surveillance reports, and (5) articles had to be published within the last 10 years (1 January 2011 to 18 February 2021).Studies were excluded if reported on: (1) non-human data, (2) non-fungal data, (3) no data on the selected criteria, (4) < 50 patients or isolates, (5) novel antifungal agents (in pre-clinical, early phase trials or not licenced), ( 6) novel diagnostic tools (not registered for routine clinical use), (7) in vitro studies of resistance mechanism(s), ( 8) case reports, conferences, abstracts, or reviews, (9) articles not written in English, and (10) articles published outside the study period.

Search strategy
PubMed and Web of Science databases were searched for possibly eligible studies published from 1 January 2011 to 18 February 2021.On PubMed, the search was optimised using the medical subject headings (MeSH) and/or keyword terms in the title/abstract for P. kudriavzevii On Web of Science, MeSH terms are not available, and therefore a topic search (TS), title search (TI), or abstract (AB) search was used.The final search used [TI = (' Candida krusei ') OR AB = (' Candida krusei ')], combined, using AND term, with criteria terms each as topic search, including (mortality) OR (case fatality) OR (morbidity) OR (hospitali * ation) OR (disability) OR (drug resistance) OR (prevention and control) OR (disease transmission) OR (diagnostic) OR (antifungal agents) OR (epidemiology) OR (surveillance).
PubMed and Web of Science databases are underpinned by a standardised taxonomy database, 8 and therefore search terms using a species name will also retrieve articles where updated or obsolete nomenclature have been used.Hence, searches using the Candida krusei term retrieved articles utilising either C. krusei or P. kudriavzevii.

Study selection
Articles searched from each database were imported into a reference manager, EndNote ®.These search results were assessed using the online systematic review software, Covidence ® (Veritas Health Innovation, Australia).Duplicate publications were removed.The remaining articles underwent title and abstract screening based on the inclusion criteria.The reasons for excluding articles were recorded during full text screening.The title/abstract screening and full text screenings were performed independently by two reviewers (HYK and SLS).Discrepancies were resolved by a third reviewer (JWA).

Data extraction
Data from the included studies were extracted for each relevant criterion by one reviewer (HYK) and independently checked by a second reviewer (JB).

Risk of bias assessment
The risk of bias assessment was independently performed by two reviewers (HYK and JB) for the included studies on relevant bias criteria, depending on the study design.Risk of bias tool for randomised trials version 2 (ROB 2) tool and Risk of bias in non-randomised studies (RoBANS) tool were used to assess the randomised controlled trials and nonrandomised trials, respectively. 9 , 10The studies were rated as low, high, or unclear risk.Each outcome criterion was assessed if any bias was expected based on the study design, data collection, or analysis in that particular study for the selected outcomes.

Data synthesis
The extracted data on the outcome criteria were quantitatively or qualitatively synthesised depending on the amount and nature of the data.Data synthesis was performed independently by two reviewers (HYK and JB).

Study selection
Overall, 818 articles were identified in PubMed ( n = 360) and the Web of Science Core Collection ( n = 458) databases.After excluding duplicated and non-relevant articles, 62 articles underwent full text screening of which 33 articles were included in the final analysis (Fig. 1 ).

Risk of bias
Of the included studies, 18 studies were classified as low risk of bias in all domains assessed (Table 1 ).Fifteen studies were classified as unclear risk of bias, due to confounding variables and selection biases caused by unclear eligibility criteria or population groups.

Inpatient care
Only one study in a tertiary care centre conducted in neonates with bloodstream infections (BSI) reported a median (IQR)

Annual incidence of infections
A prospective national surveillance study in Denmark reported an incidence of P. kudriavzevii of 0.45 per 100 000 inhabitants during 2010-2011. 27The study was conducted in 13 tertiary care centres and found a stable incidence rate of P. kudriavzevii infection from 2004 to 2011 ( ∼5% of all Candida -like blood isolates).The low incidence rate reported by this study emphasises the rarity of P. kudriavzevii infections.

Global distribution
Overall, 26 studies reported data on the distribution and emergence of P. kudriavzevii in various regions around the world (Fig. 2 ; Table S1 ).Whilst the organism is globally distributed, variations by geographic regions exist.Understanding these geographic variations is crucial for tailoring regional strategies to address P .kudriavzevii infections.Due to variable study populations, direct comparison of the distribution of P. kudriavzevii between geographic regions was challenging.Global surveillance studies reported P. kudriavzevii in 2.6% ( n = 76/2936) and 2.1% ( n = 36/1717) of cases, respectively. 28 , 29This is comparable to other studies, generally reporting a low prevalence of candidaemia due to P. kudriavzevii among Candida species in adults that ranged from 1 to 10.8%. 12 , 13 , 21 , 23 , 30-36     A high prevalence rate of 44% P. kudriavzevii candidaemia in paediatric patients in India related to an outbreak in a single year was reported. 12Environmental sources appeared to be a washbasin and genetic similarity with the environmental isolates was demonstrated.

Trends in the incidence of infections caused by P. kudriavzevii in the last 10 years
Trends in the incidence of P. kudriavzevii were variable over the last 10 years (Table S1 ).A stable incidence rate of 0-6% was reported in Japan, Denmark, Canada, and Saudi Arabia. 14 , 27 , 31 , 36A low overall incidence rate of 1.4-4.3%but with fluctuations during the period of 2011-2014 was reported in the US (2011: 4.3%, 2012: 1.4%, 2013: 4.3%, 2014: 2.1%). 13Higher incidence rates (up to 10%) were reported in France and Israel, although overall the incidence rates are decreasing from 9-10% to 2.6-3%. 21 , 37One study in India observed an increased incidence from 5.6% in 2009-2013 to 9.3% in 2014-2018. 23One study in the US in cancer patients with candidaemia reported a higher overall rate of 14-15%, which was stable during the study period of 2006-2014. 16

Discussion
Pichia kudriavzevii (C.krusei) causes severe infections in various organs and tissues including urinary , respiratory , and gastrointestinal tract and bloodstream.This can be explained by its ability to adhere to host tissue and form biofilms.By excreting proteases and phospholipases it damages the host tissue and becomes invasive.Its ability to evade the immune system and persistence in various conditions further increases the infection risk.Infections caused by P. kudriavzevii were associated with high mortality rates ranging from 44 to 67%, particularly in adults with haematologic and gastric malignancies (Table 2 ).The high 90-day all-cause mortality observed in P. kudriavzevii candidaemia likely reflects the underlying life-threatening conditions rather than the virulence of the pathogen itself. 13The association between P. kudriavzevii candidaemia and mortality is less pronounced when accounting for potential confounders such as lymphoma, neutropenia, glucocorticoid use, chronic liver disease, and elevated creatinine concentrations (HR, 1.3; 95% CI, 0.9-1.8 in multivariable analysis v er sus HR, 1.8; 95% CI, 1.3-2.4 in univariable analysis). 13This epidemiological evidence is supported by in vitro and in vivo virulence tests demonstrating that P. kudriavzevii is a relatively low-virulence pathogen, i.e., no mortality, no weight loss, no metastatic eye infections, no or discrete kidney inflammation in mice models, compared with other Candida -like species. 38The mortality rate of P. kudriavzevii is lower in paediatric compared to adult populations, which may be due to the severity of co-morbidities in adults.Numerous factors which predict mortality associated with P. kudriavzevii have been identified. 6verall, patients with immature/suppressed immune systems or imbalanced bacterial-fungal ecosystem in gut are at an increased risk of infection by P. kudriavzevii.The risk factors vary with age which include prior antibiotic/antifungal use.Antibiotics cause long-term imbalance of human gut microbiome where the eradication of certain bacteria makes room for opportunistic fungal pathogens to invade. 40 , 41Similarly, antifungals can cause bacterial-fungal imbalance in mice gut, disrupting healthy symbiotic gut flora and immune homeostasis. 424][45] As the transmission of P. kudriavzevii is common from the hands of healthcare workers and the healthcare environment, 12 , 46-48 reinforcement of hand hygiene practices and maintenance of central venous catheters has been shown to assist with infection control including for P. kudriavzevii. 23 , 49Preventative studies based on the identified risk factors should be explored for their potential benefit and feasibility for implementation to prevent P. kudriavzevii infections in these at-risk populations, including education, 50 proper antifungal prophylaxis, 51 weekly surveillance rectal swabs 52 and avoidance of unnecessary broad-spectrum antibiotics. 50he impact of P. kudriavzevii infections on length of hospital stay is poorly understood and requires further research.Clinical experience indicates that P. kudriavzevii is unlikely to cause long-term disability and secondary eye infections are rare. 39lthough P. kudriavzevii is considered intrinsically resistant to fluconazole, resistance rates to other azoles, anidulafungin and micafungin were mostly low (0-5%).Resistance to P. kudriavzevii can result from various mechanisms.Firstly, mutations in the target enzyme for ergosterol synthesis (lanosterol 14 α-demethylase, Erg11 or Cyp51) reduce the binding affin-ity of azole drugs. 53 , 54Secondly, a lower ergosterol content in the cell membrane of P. kudriavzevii can reduce the binding sites for amphotericin B, making it less effective. 54Lastly, the upregulation of efflux pumps removes drugs from the cell increasing its resistance further.The responsible efflux pump ABC1 is relevant for azole acquired resistance while the efflux pump ABC2 is associated with innate resistance to fluconazole. 55 , 568][59] Resistance rate to caspofungin, i.e., the proportion of isolates with MICs above the CLSI breakpoint 60 (EUCAST breakpoint has not been determined because of high variation in caspofungin MICs), 61 has varied in the included studies.MIC testing for caspofungin is considered unreliable/non-reproducible by both CLSI and EUCAST. 61Whilst most studies reported relatively low non-WT rates of 0-6%, three studies reported higher non-WT rates of 30-67%. 19 , 21 , 22The high non-WT rates to caspofungin are likely misclassified as (1) applying the CLSI breakpoint to varying MICs might lead to falsely reporting too many wild-type strains as non-susceptible; 19 , 22 , 61 (2) combining the CLSI breakpoint with E-tests has not been validated, 21 , 60 , 62 and (3) higher MIC ranges obtained by Etests than by CLSI method that might lead to 67% of the cases being misclassified. 38In waiting for validated methods for testing caspofungin, EUCAST recommends using anidulafungin or micafungin as predictors of resistance for echinocandins. 61Similarly, reduced susceptibility to amphotericin B has also been observed, with the proportion of isolates with MICs > CLSI ECV 17 being reported in India (13%) and Iran (40%). 19 , 23The reduced susceptibility might be explained the fact that amphotericin B is one of the most common antifungal drugs used in these regions. 19It is likely that there will be geographic variability of MICs and it is vital that testing laboratories in LMICs always utilise quality control strains to ensure their results are in accord with international standards. 63National and/or international surveillance systems are required to systematically monitor the development of resistance for P. kudriavzevii.Data from these systems would support clinicians in making decisions based on information from their local region including epidemiology, antimicrobial resistance, and treatment strategies.In addition, appropriate use of antifungal drugs promoted by timely, accurate diagnosis and susceptibility testing will assist in reducing the risk of resistance development. 64To reduce the high mortality associated with P. kudriavzevii , traditional phenotypic methods (e.g., colony morphology, biochemical tests or Analytical Profile Index -API) are useful for screening P. kudriavzevii to initiate echinocandin or amphotericin B instead of fluconazole.However, specialised methods such as DNA sequencing or MALDI-TOF mass spectrometry provide more accurate and reliable species identification.Indeed, nine studies evaluating the accuracy of different methods for the identification of uncommon Candida species including P. kudriavzevii found that the accuracy using traditional phenotypic methods ranged from 15-76% versus 75-100% for MALDI-TOF MS or sequencing. 65 , 66Higher accuracy when using traditional methods was achieved by colonising P. kudriavzevii on CHROMagar medium and incorporating a specific screening test for P. kudriavzevii (e.g., immunoassay Krusei-Color Fumouze ®) in replacement with API system. 67 , 68Regionalor country-specific treatment strategies should also be developed due to the diverse P. kudriavzevii resistance pattern and resources available. 63New drugs like rezafungin, 69 ibrexafungerp 70 and oteseconazole 71 will likely be a valuable addition to the therapeutic armamentarium to treat P. kudriavzevii infections as they have demonstrated activity against fluconazole resistant isolates.
The incidence of P. kudriavzevii, although globally distributed, varies depending on the population studied and geographical location.Generally, the incidence of P. kudriavzevii was low and stable across the globe except for India where the incidence has been increasing over the last 5 years (5.6-9.3%).The increased trend in India may be due to high hospital occupancy and challenges in infection control implementation leading to cross-transmission. 11It is also possible that incidence rates of P. kudriavzevii in LMICs are underestimated due to difficulties in species-level identification in the absence of mass spectrometry or molecular techniques in routine clinical practice. 72 , 73e acknowledge several limitations in our review.First, publication bias cannot be excluded as few observational studies on the incidence and clinical outcomes of P. kudriavzevii infections and laboratory-based studies for susceptibility data from LMICs were retrieved by our search.Studies from under-resourced settings may be smaller scale due to limited financial and human resources, leading to publication bias in favour of better resourced settings. 74Second, many studies were retrospective cohort studies where selection bias might have occurred and there might have been an absence of data on potential confounders.Third, language bias cannot be ruled out as we only searched English language literature.Considering these limitations, we interpreted the results cautiously.Although studies published before 2011 were excluded, the outcome criteria assessed are time-sensitive rendering older data less informative.

Conclusion
Mortality in patients with P. kudriavzevii (C.krusei) infection was higher for adults than paediatric populations, particularly those with severe co-morbidities.Rapid identification of P. kudriavzevii is vital to administering echinocandins or high doses of amphotericin B as initial treatment.Non-WT rates of azoles and echinocandins was low, except for fluconazole.Risk factors for developing P. kudriavzevii infections vary with age, notably low birth weight, prior use of antibiotics/antifungals, and an underlying diagnosis of gastrointestinal disease or cancer.The implementation of stewardship programmes focused on addressing these risk factors should be explored for their benefit and feasibility.Although rare, P. kudriavzevii is globally distributed with an apparently higher incidence in India.This highlights the need for continued surveillance efforts and targeted interventions to address P .kudriavzevii infections, particularly in regions with higher incidence rates.Due to scarce data on incidence and resistance, stronger and global surveillance systems are required to support clinical decisionmaking for P. kudriavzevii.

A c kno wledg ements
This work, and the original report entitled 'WHO Fungal Priority Pathogens List to Guide Research, Development, and Public Health Action', was supported by funding kindly provided by the Governments of Austria and Germany (Ministry of Education and Science).We acknowledge all members of (C. krusei) and criterion.The final search used was ( C. krusei [Title/Abstract]) combined, using AND term, with criteria terms including (mortality [MeSH Terms]) OR (morbidity [MeSH Terms]) OR (hospitalisation [MeSH Terms]) OR (disability[All Fields])) OR (drug resistance, fungal[MeSH Terms]) OR (prevention and control[MeSH Subheading]) OR (disease transmission, infectious[MeSH Terms]) OR (diagnostic[Title/Abstract]) OR (antifungal agents[MeSH T erms]) OR (epidemiology[MeSH T erms]) OR (surveillance [Title/Abstract]).

Figure 1 .
Figure 1.PRISMA flow diagram for selection of studies included in the systematic review of P. kudriavzevii.Based on: Preferred Reporting Items for Sy stematic R e vie ws and Meta-Analy ses: T he PRISMA Statement.

Table 1 .
Risk of bias of included studies.

Table 2 .
Mort alit y associated with P. kudria vz e vii.

Table 3 .
Studies reporting drug susceptibility of P. kudria vz e vii.

Table 4 .
Drug susceptibility of P. kudria vz e vii to azoles.

Table 5 .
Drug susceptibility of P. kudria vz e vii to non-azole antifungal drugs.

Table 6 .
Risk factors for infections caused by P. kudriavzevii.